TWI225490B

TWI225490B - Bioactive whey protein hydrolysate

Info

Publication number: TWI225490B
Application number: TW088110663A
Authority: TW
Inventors: Ralf-Christian Schlothauer; Linda May Schollum; Anne Maria Singh; Julian Robert Reid
Original assignee: New Zealand Dairy Board
Priority date: 1998-06-17
Filing date: 1999-06-24
Publication date: 2004-12-21
Also published as: WO1999065326A1; DE69920219D1; DK1087668T3; EP1087668B1; NO20006305L; US7148034B2; DE69920219T2; US20090258387A1; US6919314B1; AU761477B2; PE20000717A1; ATE275831T1; NO20006305D0; EP1087668A1; AU4535999A; NO320644B1; NZ508867A; US20040086958A1; US20050164340A1; MY126517A

Abstract

The invention relates to a partial hydrolysate of whey protein which contains bioactive peptides but does not have a bitter flavour. The hydrolysate is carried out using selective enzymes which produce the active peptides and is terminated at a degree of hydrolysis before substantial bitter flavours are created. There are also described novel peptides and a method of reducing systolic blood pressure through the administration of the peptides.

Description

技術領域：本發明係關於一種萝-不呈^ 1 Γ ——〜η 肽類之到生恭ώ u 不，、古味且含有具丨生物if性丨之艇肢類之礼α蛋白巧水解物的方法。此方氺Ϊ 一古玄 η ^ ^21 V- Ah 1, 凡万法之產物有呵 >為化率及良好的\ g月色性。此箄產物可处曰ϋ—m ι 寺座物了犯具有溫和或些微的甜味，窠醣,I Α ^未此寻礼4蛋白水解物可視需要含有來源。、铖此性艮口口中之生物活性胜肽類之有致背景技藝許多食品成份及食物已經是乳蛋白，酪蛋白及乳清蛋白等在健康照護的許多領域中，水解蛋白質食物之優勢。舉例白質較不具過敏原性。其亦能吸收。含有水解蛋白質之食物患者之攝食也有助益。由水解蛋白質來源，如：牛而製成。水解蛋白質食物具有超越非而言，已知經酵素水解之蛋車父全蛋白更快速地被消化及對醫院病患如具消化性疾病Technical Field: The present invention relates to a kind of glutinous-non-presenting ^ 1 Γ ～~ η peptides. No, antique, and containing 丨 bio-if 丨 boat polite gift α protein hydrolysate Methods. This side is an ancient mystery η ^ ^ 21 V- Ah 1, the product of Fanwanfa has a chemical conversion rate and good moonlight properties. This product can be described as "ϋ"-m ι temple seat has a mild or slightly sweet taste, caramel, I ^ ^ ^ this protein 4 hydrolysate may contain the source as required. This is because of the biologically active peptides in the mouth. Background technology Many food ingredients and foods are already milk protein, casein, and whey protein. In many areas of health care, hydrolyzed protein foods have the advantage. Examples White matter is less allergenic. It can also be absorbed. Foods containing hydrolyzed protein Food intake by patients also helps. Made from hydrolyzed protein sources such as cattle. Hydrolyzed protein food has more than that. For example, the enzyme-hydrolyzed egg is known to be digested more quickly by whole car protein.

已知將乳清蛋白及酪蛋白之水解可釋出呈現數種生理效用的生物活性胜肽類（Maub〇is等人，1991，歐洲專利第 4755 0 6 )號。許多刊物述及此等具生物活性之胜肽類，例如·經由酵素處理之牛血清沒—乳球蛋白及乳清蛋白濃縮物而得到之具有抗高血壓性之ACE抑制胜肽（MuUally等人，1 997 )。ACE抑制胜肽亦發現存在於αg及召—酪蛋白之水解物中（日本專利第4282400號；Nakamura等人，1 994， Yamamoto 等人，1994)。歐洲專利第4745506號揭示乳清中之牛乳乳鐵蛋白水解It is known that hydrolysis of whey protein and casein can release bioactive peptides exhibiting several physiological effects (Maubois et al., 1991, European Patent No. 4755506). Many publications mention these biologically active peptides, such as the anti-hypertensive ACE inhibitory peptides obtained from enzyme-treated bovine serum-lactoglobulin and whey protein concentrates (MuUally et al. , 1 997). ACE-inhibitory peptides are also found in hydrolysates of αg and tau-casein (Japanese Patent No. 4282400; Nakamura et al., 1 994, Yamamoto et al., 1994). European Patent No. 4745506 discloses hydrolysis of bovine milk lactoferrin in whey

第4頁五發明說明（2) 可以釋出乳鐵蛋白冬， ’舄’、香港腳’眼部感染，；l :當；：治療人類或動物之腹然而，已知大部份、決病之抗微生物劑。The fifth page of the invention description (2) can release lactoferrin winter, '舄', Hong Kong feet 'eye infections; l: when ;: treatment of the abdomen of humans or animals Antimicrobial agent.

Si產：之水解解^其是含有乳清及 =口以蛋白水解物⑵=: f ^白貝水解之領域中，通常使一 =來控制或去除蛋白f水解物：種或同時使用兩種味性。未，以增加產品的美已知廣泛地水解蛋白質基質可味（歐洲專利第0 65663 ’1 1 7047號，美礼蛋白水解物之苦號）。此法可較簡易且較便宜地生產‘”第39·0 而，廣泛地水解會輪短所有胜狀 -古味之產品。然具有生物活性之胜肽。廣泛地水：質=::得到之得到之胜肽的機能性及生物活性。此外貝欲質基質之溫和味道仍較差相=來蛋白而f ’苦味只部份被去除（Roy 1992及1 9 9 7 )。t水解物味【音種：t控！1蛋白質水解物苦味的方法是利用去苦者’、，、是彼等來自米麴菌（Aspergillus 〇ryzae) ，蛋白質水解所產生之”苦味”認為係肇因於大疏水性，，苦 "f 胜肽之存在。去苦味酵素可選擇性地水解存在於蛋白質水解物中之苦味胜肽。熟習此技術之工作者可藉由明智Produced by Si: Hydrolyzed ^ It contains whey and = protein hydrolysate ⑵ =: f ^ in the field of white shell hydrolysate, usually use a = to control or remove protein f hydrolysate: species or use both Flavor. In order to increase the beauty of the product, it is known to extensively hydrolyze the taste of the protein matrix (European Patent No. 0 65663'1 1 7047, Merlot protein hydrolysate). This method can be simpler and cheaper to produce "" Article 39 ·. Extensive hydrolysis will shorten all the products of the vicissitude-antique taste. However, it has biologically active peptides. Extensive water: quality = :: obtained The functional and biological activity of the obtained peptide. In addition, the mild taste of the shellfish matrix is still poor. Phase = protein and f 'bitterness is only partially removed (Roy 1992 and 197 7). Species: t control! 1 The method for bitterness of protein hydrolysates is to use bitters, ',, and they are from Aspergillus 〇ryzae. The "bitterness" produced by protein hydrolysis is thought to be caused by large hydrophobicity. The existence of bitter peptides. Debittering enzymes can selectively hydrolyze bitter peptides present in protein hydrolysates. Workers familiar with this technology can use wise

第5頁 1225490 發明說明（3) =及處理條件，將牛乳蛋白水解物中之苦 _ :然"；留下所欲得到具生物活性之胜且斗仏X ^ 使用去古味酵素使得製程更加昂貴，且一些得。Η = = Τ來或被成功地獲酸至終產物之趨向，結果具有釋出游離胺基味或4味。UQy 1 992及1 997 ) 7 生令人*悅之肉湯各式各樣的去除蛋白水解額外製造步驟及增加势送=古2方法均導致終產物之酸終產物變成超出平；？成本…卜，由於產生游離胺基倘若能開發出一種水組I ^I 物活性之胜肽類耧ψ B蛋白貝之方法，使所欲得到具生成，可限制苦味胜狀及游離胺基酸生大之優Ϊ 質之溫和味叇得以保冑，則將具有莫一些具生物活性之胜肽類 ^ 質水解過程中相對地較為安定且非常二血壓胜肽-在蛋白之水解過程中釋放出來(如圖…』）：易於牛乳蛋白基質牛乳蛋白水解物的苦味可藉由添加牛乳蛋白基質中之天然糖類( ^員或水解已存在於 (Β⑽amlen，1 98 9 )。例如 ^主而加以改善 ^ 一半乳糖甘酶及乳糖鎢將乳J = = =清若藉其更具美味性（法國專利第2309 1 54號；m匕，可使 4358464號；日本專利第㈣咐^號）。關專利第為了使含有具生物活性之胜肽類的蛋白質水解產 1225490Page 5 1225490 Description of the invention (3) = and processing conditions, the bitterness in the milk protein hydrolysate _: Ran " leave the desired biologically active victory and 仏仏 X ^ use of anti-palatase enzyme to make the process more Expensive and some too. Η = = T tends to be or is successful in obtaining the acid to the final product, with the result that it has a free amine or 4 flavor. UQy 1 992 and 1 997) 7 Delightful broths of all kinds Remove proteolysis Extra manufacturing steps and increase potential = ancient 2 methods have all caused the acid end products of the final products to become more than flat; Cost ... By, because the production of free amine groups can be developed if a peptide of the water group I ^ I activity is active 耧 ψ B protein shell, so that the desired production can be achieved, the bitter taste and free amino acids can be limited Shengda Zhiyou, the mild miso of quality can be preserved, then some peptides with biological activity will be relatively stable ^ during the hydrolysis of the protein is relatively stable and very ditensive peptides-released during the hydrolysis of the protein (As shown in the picture ...): The bitter taste of cow milk protein hydrolysate which is susceptible to the milk protein matrix can be improved by adding natural carbohydrates in the cow milk protein matrix (^ members or hydrolysis is already present (B⑽amlen, 1 98 9). ^ Galactosidase and Tungsten Lactose J = = = Qing Ruo will be more delicious (French Patent No. 2309 1 54; m dagger can make No. 4358464; Japanese Patent No. ^). Related patents The first is to hydrolyze proteins containing bioactive peptides to produce 1225490.

五、發明說明（4) 高度的風味可接受性，有必要精確地控制水解過程以避免苦味生成。一種常用來終止水解反應的方法是藉由將酵素去活性化，通常在高溫藉熱去活化，於9 0〜丨0 〇〇c以上維持/一段時間。然而，此方法無法用來終止乳清蛋白之水解，'因^ ^ 在於混合物中之完全未被水解的乳清蛋白會而變性且沉殿，使得終產物之溶解度較差且其用來作為食品成份2接受度亦較牴。倘若有一種製造水解乳清蛋白質之方法，可經控制使得直接生成含有生物活性胜狀類以供添加於機能性食品之水解物，其不具苦味且酵素之去活性化步驟不會破壞Z產物中所含之完整蛋白質，將會是有利的。本兔明之一目的即疋提出一些有法，以期逵到此蓉兩求，或者，至少提供大眾一個有用的選擇？達到此寺而發明概述因此，本發明可廣泛地述及製備含有生物活性胜肽之乳清蛋白水解物的方法，其包括以下步驟： (i )以一或多種可水解乳清蛋白之酵素處理乳清蛋白基質，以生產一種含有生物活性胜肽之乳清蛋白水解物；及 (i i)於苦味風味實質生成之前終止水解反應。 &較佳地將水解作用在最多只造成部份乳清蛋白變性且仍能維持或改善此水解物之官能性的反應條件下終止。更佳地’水解作用能在部份乳清蛋白變盖官能性的反應條件下被終止。。一5. Description of the invention (4) High flavor acceptability, it is necessary to precisely control the hydrolysis process to avoid bitterness. A commonly used method to stop the hydrolysis reaction is to deactivate the enzyme, usually by deactivating it at high temperature, and maintaining it for more than 90 ° C to 0 ° C for a period of time. However, this method cannot be used to stop the hydrolysis of whey protein. 'Because the completely unhydrolyzed whey protein in the mixture will denature and sink, making the final product poorly soluble and used as a food ingredient 2 Acceptance is also relatively low. If there is a method for producing hydrolyzed whey protein, it can be controlled to directly generate a hydrolysate containing biologically active species for addition to functional foods, which does not have a bitter taste and the enzyme deactivation step does not destroy the Z product. The intact protein it contains will be beneficial. One of the goals of this rabbit is to propose some ways to achieve this, or at least provide a useful option for the public? Summary of the invention upon reaching this temple Therefore, the present invention can broadly describe a method for preparing a whey protein hydrolysate containing a biologically active peptide, which includes the following steps: (i) treatment with one or more enzymes that hydrolyze whey protein A whey protein matrix to produce a whey protein hydrolysate containing a bioactive peptide; and (ii) stopping the hydrolysis reaction before the bitter flavor is substantially generated. & The hydrolysis is preferably terminated under reaction conditions which at most cause denaturation of part of the whey protein and still maintain or improve the functionality of this hydrolysate. More preferably, the 'hydrolysis can be terminated under a reaction condition where part of the whey protein becomes functional. . One

第7頁 1225490 五、發明說明（5) 較佳地，用以水解乳清蛋白的酵素可選自於下列所組之群：蛋白酶P6，蛋白酶A，蛋白酶M，胜肽酶，中性酶’有效酶（Validase)及AFP 200.0 (均如本文所定水解之終止以熱處理較佳，處理時間較佳以i至1〇秒，在溫度約8 5 °C至1 0 0 °C。水解終止前之基質水解程度以至多丨〇%較佳。水解程度以約3%至約5%更佳。基質以含乳糖達50%重量比者較佳。或基質中含乳糖達3 0 %者亦可。、基質以經由乳糖酶及/或点-半乳糖苷酶處理者較佳， i者ΐ於乳清蛋白水解前或水解期間，將乳糖水解成半朝糖及葡萄糖並合成半乳募糖。本=明之另一較佳具體實施㈣是一種乳清蛋白水解 AFFΓ或多種生物活性胜肽，選自下列所組成之群： VTSnv ^ ’ IUEKH，UVTQ，MKG，LDIQK，VF，ALP_， vts^av ，lhlplp ，LVYPFPGPIPNSLPQNIpp及LFRQ 。之理nn:藉由熟習此等技術的人對某種特定酵辛之=解’而於適合該特定酵素之條件下進行。阵素 :用於乳清蛋白濃度為：固形物5〜5〇% 總固形广為"酵素二基質比介於。.。崎。(重量/ 重量比）者^佳比）其中以介於〇.〇1%至1%(重量/總固形物性蛋白酶處理蛋白質基質，可於ρΗ 25 進仃水解，並以ρΗ 3.0〜5.〇之間較佳。之間Page 7 1225490 V. Description of the invention (5) Preferably, the enzyme used to hydrolyze whey protein may be selected from the group consisting of protease P6, protease A, protease M, peptidase, neutral enzyme ' Validase and AFP 200.0 (both as stated herein, the termination of hydrolysis is better by heat treatment, the treatment time is preferably from 1 to 10 seconds, at a temperature of about 8 5 ° C to 100 ° C. Before the termination of hydrolysis The degree of matrix hydrolysis is preferably at most 10%. The degree of hydrolysis is preferably about 3% to about 5%. The matrix is preferably 50% by weight with lactose or the matrix contains 30% lactose. The substrate is preferably treated with lactase and / or dot-galactosidase, and those who have hydrolyzed lactose to hemichalcose and glucose and synthesize galactose before or during the hydrolysis of whey protein. = Another preferred embodiment of Ming is a whey protein hydrolyzed AFFΓ or multiple biologically active peptides selected from the group consisting of: VTSnv ^ 'IUEKH, UVTQ, MKG, LDIQK, VF, ALP_, vts ^ av, lhlplp, LVYPFPGPIPNSLPQNIpp, and LFRQ. nn: People who are familiar with these technologies The specific enzyme is the solution. It is carried out under the conditions suitable for the specific enzyme. Matrix: used for whey protein concentration: solids 5 ~ 50% The total solids is widely known as the enzyme two matrix ratio .. . Saki. (Weight / weight ratio) is better.) Among them, the protein matrix is treated with 0.001% to 1% (weight / total solid physical protease), which can be hydrolyzed at ρ 25 and ρ 3.0 3.0 ~ It is better between 5.

第8頁 1225490 五、發明說明（6) 若以中性蛋白酶處理蛋白質基質，可於ρΗ 3 · 5〜9 . 〇之間進行水解，並以pH 6· 0〜8· 0之間較佳。若以鹼性蛋白酶處理蛋白質基質，可於pH 5· 0〜10. 〇之間進行水解，並以p Η 6 · 0〜8 · 0之間較佳。蛋白質水解可於溫度範圍20-65 °C下進行，並以50-60 °C 較佳。乳糖水解之進行可進行於乳清蛋白水解之先前階段，同時ί ^後。用來水解乳糖的酵素可為乳糖酶及/或冷—半乳 ^ It 且其可遥自酵母或真菌來源，如：Klyvermyces Saccharomyces lactis, Saccharomyces fragillls，如米麴菌，黑麴菌，如 des)及Novolact(Novc) Nordisk)。乳糖水解於孰習此技術之人員所知之條件下進行+ …、一種終止水解方、、土一蛋白水解酵素式此：：貫施例乃藉由使一或多種乳清化來達成，首弁获2先刖加入之乳糖水解酵素）之去活性失活或較不具活=將反應混合物之酸鹼值改變至使酵素合物加熱至幸交、^ 4之酸鹼值，及/或用熱交換器將反應混之完整乳清蛋白變里度，以使酵素變性但卻不使基質中 70 °c，較佳為65。7。。酵素失活的適當溫度範圍為55- 根據一選擇，與所燥之步驟使酵素用之酵素有關，亦可使用蒸發或乾根據另一選擇，素混合物失活。整酸鹼值而去活化酵素或酵素混合物可以先前調整或不調Page 8 1225490 V. Description of the invention (6) If the protein matrix is treated with a neutral protease, it can be hydrolyzed between ρΗ 3 · 5 ~ 9. 〇, and preferably between pH 6 · 0 ~ 8 · 0. If the protein matrix is treated with alkaline protease, it can be hydrolyzed between pH 5.0 · 10 ~ 1.0, and preferably between pΗ6 · 0 ~ 8 · 0. Proteolysis can be performed at a temperature range of 20-65 ° C, and preferably 50-60 ° C. The lactose hydrolysis can be carried out at a previous stage of whey protein hydrolysis, and at the same time. The enzymes used to hydrolyze lactose can be lactase and / or cold-galactose ^ It and it can be remote from yeast or fungal sources, such as: Klyvermyces Saccharomyces lactis, Saccharomyces fragillls, such as Oryzae, black fungus, such as des) And Novolact (Novc) Nordisk). Lactose hydrolysis is carried out under conditions known to those skilled in the art +, a method for terminating hydrolysis, and a protein-hydrolyzing enzyme formula: This example is achieved by making one or more whey, first (Get 2 lactose hydrolyzed enzymes added before) Deactivation inactivation or less active = change the pH value of the reaction mixture to heat the enzyme compound to Xingjiao, pH value of 4 and / or use The heat exchanger mixes the reaction with intact whey protein to denature the enzyme without denying 70 ° C in the matrix, preferably 65.7. . The appropriate temperature range for enzyme inactivation is 55- depending on the choice, depending on the enzyme used in the drying step, or by evaporation or drying. According to another choice, the inactivation of the vegetarian mixture. Adjust pH or deactivate enzymes or enzyme mixtures can be adjusted previously or not

第9頁 1225490 五、發明說明（7) —或者，用以選擇性水解乳清蛋白的一種或 ::二ΐίΓϊ’如：RoehmEupergit，鹿角Ϊ“ 粒，成丁聚醣顆粒或任何其它合適的物質上=:膠顆器。膜上，或在巾空纖維管反應此素可於水解作用進行前，A 方式固定於適當的鈍性支持物上 ^鏈、之濾膜加以分離。 1γ κ解作用後以微過或者，酵素可於水解完成時，立 1 〇- 5 0 0仟道耳頓（KDa)之超過滹/ :/慮過分子量來。愿朕自水解混合物中分離出在水解及視需要使酵素失活或移物可視需要在條件下進行逆滲透萨此自j，此等水解類及水份。如此便可回收含有乳清‘白除鹽水解物中還會含有葡萄糖，=;;將:;唐加，水解，則含有具生物活性之胜肽類的乳清 3水解< I糖。使用可遽過分子量5_200仟冑耳頓^^水^物亦可視需要較佳為10-50仟道耳頓之。具生物活=分離出來，肽類及，視需要地，被水解的乳糖丨肽類，其他胜收。胖扪礼搪均可於過濾液中被回根據另一選擇，離子交換樹脂或互作用層析法或此等方法之組合亦；於=或疏水*** 而得到較強化型式的具生物活性部份。；7解物中回收Page 9 1225490 V. Description of the invention (7)-Or, one or selectively used to selectively hydrolyze whey protein, such as: "Roehm Eupergit, Staghorn", granules, into butanose particles or any other suitable substance Top =: gel particles. On the membrane, or before the reaction of the hollow fiber tube, the element can be fixed on the appropriate blunt support ^ chain, and the membrane is separated before the hydrolysis. 1γ κ solution After that, the enzyme may be passed by when the hydrolysis is completed, and the molecular weight will be more than 10/500 (Da) (KDa), and the molecular weight will be taken into consideration. Need to make the enzyme inactivated or move the material under the conditions of reverse osmosis, such as j, such hydrolysis and water. So you can recover whey 'white desalted hydrolysate will also contain glucose, =; Will be: Tanga, hydrolyzed, whey 3 containing bioactive peptides hydrolyzed < I sugar. The use of a molecular weight of 5 to 200 仟胄仟胄 ^ ^ water ^ may also be better if necessary 10 -50 仟 Doorton. With biological activity = isolated, peptides and, if necessary Hydrolyzed lactose 丨 peptides, others win. Fat can be returned in the filtration solution. According to another option, ion exchange resin or interaction chromatography or a combination of these methods is also available; in = or hydrophobic Sexual intercourse to obtain a stronger type of biologically active part .; 7 solution recovered

O:\58\58905.ptd 第10頁 1225490 五發明說明（8) __ 以乳糖…-半乳糖苔酶水解： +礼券糖已知其可促進腸内有益菌之生斤產生之水解物之生物活性特性。、匕亦可提高進-步水解乳糖水解物對乳糖者而έ ，是很有用的食品添加物。了足之4費本發明之水解乳清蛋白產品具有—或 • t =壓性之升壓素轉換酵素抑制劑⑽」ΪΓ •雙歧桿菌生長促進活性 11 •不黏，不具苦味 • 具有悅人的至輕微的甜味 •良好的官能性二！：包括上述之說明及後述之實例在内。圖式間要說明一現在將對本發明之附圖進行說明，其中. 圖1是一以苦味及生物活性為縱軸/以水 =圖。根據本發明而於苦味胜肽類生成之前庐王二為橫軸活性之胜肽類並具有可接受性二:：有具生於線\及線x2之間。禾之產的機會窗"介表示四組以不同飼料餵食8週之期血壓變化圖。大白以的收縮银Γ八為么曰以餵食商品化大白鼠飼料為控制組，相料均值分析圖。的幻枓之大白鼠的最小平方又义口口 . 1225490 五、發明說明（9) 如刖文所述，本發明提供一之乳清蛋白水解物之方法，苴衣^ 3生物活性胜肽類 π避免在水解期間產生；;良解如作用=高度^制下 =。水解作用如圖i所示，於 =，及即於大量苦味生成前即終止中被終止’ 最大量之生物活性胜肽類：供：2m性及於圖1中之X軸。介於赴v sa K解私度定Ϊ地表示素而變動。最適水解條件即\曰使的苦機會窗將^ 生物活性達最高量。未在了接文之範圍内而酵ΐίΓ=;!Τ圭具體實 <列，水解乳清蛋白所用的坪系你&自下列所組成之群：白， ^ 醜’胜肽酶’”生酶，有效酶及AFP2_(=本= 疋，且藉由南溫短時之熱處理X約85- 1 0 0。〇處理卜！〇秒）終止水解作用。於此應用中可喜地發現上述酵素（1 生產含不少量生物活性胜肽類之乳清蛋白水解物（2)可於短時之熱處理下失活，處理過程中只有部分乳清蛋白吏、’且令人驚喜地改善乳清蛋白之官能性，包括提供具有滑潤口感（因其顆粒相對較小）且外觀相當潔白。八” 現在將本發明以下列實例舉例說明之實例1 蛋白質含量為80%之甜乳清蛋白濃縮物之1〇%溶液 ULACENTM3 92，2公升），於50 °C下，以由枯草桿菌生產之商品化酵素：中性酶（N〇v〇 N〇rdisk，丹麥）水解，水解O: \ 58 \ 58905.ptd Page 10 1225490 Five descriptions of the invention (8) __ Hydrolysis with lactose ...- galactosidase: + Gift coupon sugar is known to promote the hydrolysate production of beneficial bacteria in the intestine Active properties. It can also improve the further hydrolysis of lactose hydrolysate, and it is a very useful food additive. The full cost of the hydrolyzed whey protein product of the present invention is-or t = compressive vasopressin-converting enzyme inhibitor ⑽ "ΪΓ • bifidobacterium growth promotion activity 11 • non-sticky, no bitter taste • pleasant To slight sweetness • Good functionality two! : Including the above description and examples described later. The drawings will be explained in the following. The drawings of the present invention will now be described. Among them, FIG. 1 is a graph with bitterness and biological activity as the vertical axis / water = graph. According to the present invention, before the generation of bitter peptides, Luwang II is a peptide with horizontal axis activity and is acceptable. 2: It is born between line \ and line x2. The window of opportunity of He Zhichan said that the blood pressure changes of the four groups were fed with different feeds for 8 weeks. The shrinkage of Dabai is based on the analysis of the mean value of the average data of the feedstock of commercial rats as the control group. The least square of the phantom of the rat is 1225490 V. Description of the invention (9) As described in the text, the present invention provides a method of hydrolysate of whey protein, 苴衣 ^ 3 biologically active peptides π avoids production during hydrolysis; good solutions such as action = height ^ system under =. The hydrolysis is shown in Figure i, at =, and is terminated before a large amount of bitterness is generated. The maximum amount of biologically active peptides: for: 2m, and on the X axis in FIG. It varies depending on the expression of v sa K. The most suitable hydrolysis conditions are the window of opportunity for suffering and the highest biological activity. It is not within the scope of the text to leave the ΐΓ = ;! Τ 圭 concrete implementation list, which is used to hydrolyze whey protein. The group consists of the following: white, ^ ugly 'peptidase' Enzymes, effective enzymes and AFP2_ (= this = 疋, and by the short-term heat treatment of South temperature X about 85-1 0 0. 0 treatment Bu! 0 seconds) to terminate the hydrolysis. In this application, the above enzyme can be found with joy (1 Production of whey protein hydrolysates containing not a small amount of bioactive peptides (2) can be inactivated by short-term heat treatment, only part of the whey protein is processed, and the whey protein is surprisingly improved Functionality, including providing a smooth mouthfeel (because of its relatively small particles) and a fairly white appearance. Eight "Example 1 of the present invention is exemplified by the following examples: 1 of a sweet whey protein concentrate with a protein content of 80% 〇% solution ULACENTM3 92, 2 liters), at 50 ° C, hydrolyzed with a commercial enzyme produced by Bacillus subtilis: neutral enzyme (NovvNordisk, Denmark), hydrolyzed

O:\58\58905.ptd 第12頁 1225490 五、發明說明（10) 水解物之酸鹼值調為5· 〇並於65 t加熱3〇分鐘以使酵素失 =°續將水解物（水解度為4· 5%)加以噴霧乾燥並測試其升壓素轉換酵素抑制劑（ACE- I)活性及風味。乾燥產品之 ACE-Ι 活性根據Dw Cushman 及HS Cheung (1971)之方法，以FAPGG(產品305-1〇， Sigma Chemical Corporation ，O: \ 58 \ 58905.ptd Page 12 1225490 V. Description of the invention (10) The pH value of the hydrolysate is adjusted to 5.0 and heated at 65 t for 30 minutes to lose the enzyme = ° (4.5%), spray-dried and tested for its vasopressin-converting enzyme inhibitor (ACE-I) activity and flavor. The ACE-I activity of the dried product was determined according to the method of Dw Cushman and HS Cheung (1971) with FAPGG (product 305-1, Sigma Chemical Corporation,

St· Louis ’ USA)為基質測定之。其活性以使ACE_i活性降低一半所需之物質量（公克/公升）表示之。此水解物之IC5q ACE-I活性為〇· 44公克/公升且經味覺試驗而測得之風味接受度分數相當高。實例2 ^ ALACENTM 421乳清蛋白濃縮物之50%溶液（蛋白質含量 56% 公升以由Kluveromyces lactis生產之商品化酵素乳糖酶（Lactozyme 3000L，Novo Nordisk)於酵素 / 基貝比為0 · 3 /，5 〇 C下，處理2小時。再將此溶液以中性酶 · 於酵素/基質比為〇· 3%，50 °C下，水解1小時。之後，將混合物稀釋五倍’藉超高溫瞬間加熱處理（95 °C，5秒）使活 -性^素失活。續將該水解物喷霧乾燥。乾燥粉末（水解度 2· 8%)並不含任何活性酵素且具有ACE-Ι活性為2· 18公克/ f升。其風味接受度分數因產品受到低度之甜化而例外地南。ACE- I測定及風味接受度之評分方法同實例1。〇實例3 以與貫例1相似之方法，由ALACENTM 3 9 2製得之5 0 0公升水解物於酵素失活後，冷卻至1 0 °C。將原始水解物取出一部伤加以乾燥’剩餘水解物在1 〇 °C以能截切分子量1 0，〇〇〇St. Louis' USA) was determined by matrix. Its activity is expressed in terms of the mass (g / litre) required to halve the activity of ACE_i. The IC5q ACE-I activity of this hydrolysate was 0.44 g / liter and the taste acceptance score measured by a taste test was quite high. Example 2 ^ 50% solution of ALACENTM 421 whey protein concentrate (56% liters of protein with a commercial enzyme lactase (Lactozyme 3000L, Novo Nordisk) produced by Kluveromyces lactis at an enzyme / kibe ratio of 0.3 /, Treatment at 50 ° C for 2 hours. This solution was then hydrolyzed with a neutral enzyme · in enzyme / matrix ratio of 0.3% at 50 ° C for 1 hour. After that, the mixture was diluted five times' by ultra-high temperature instant Heat treatment (95 ° C, 5 seconds) deactivates the active-active hormone. The hydrolysate is spray-dried. The dry powder (degree of hydrolysis 2.8%) does not contain any active enzymes and has ACE-1 activity. It is 2.18 grams / f liter. Its flavor acceptance score is exceptionally low due to the low degree of sweetening of the product. The ACE-I measurement and flavor acceptance score method is the same as in Example 1. 〇Example 3 is the same as in Example 1. In a similar way, 500 liters of hydrolysate made from ALACENTM 3 92 was cooled to 10 ° C after the enzyme was deactivated. The original hydrolysate was taken out and wounded, and the remaining hydrolysate was at 10 ° C. In order to cut the molecular weight 10,000

第13頁 1225490 五、發明說明（11) 道耳頓之過渡膜（HFK 131，Koch Membrane Systems， USA)進行超過濾。該水解物（水解度介於3 · 8%至4· 2%之間）被濃縮至V C F 1 0且濃縮液直接進行乾燥。濾過液以蒸發法濃縮至2 5 %固形物並加以乾燥。A C Ε - I測定及風味接受度之評分係如實例1測定。濾過液粉末之ACE- I活性提高（濾過液粉末之I Cm為0 · 1 5公克/公升）。於超過濾之前取出之原始水解物，經乾燥後之ACE-Ι活性為〇· 43公克/公升。濃縮液乾燥粉及噴霧乾燥粉之風味接受度均高。實例4 得自 ALACEFM 392，ALACE 評 421 及 ALACENTM 392 和乳糖 φ 之混合物之三種不同溶液被製成至多為丨5 %固形物水產生 150升。以由枯草桿菌生產之商品化酵素：中性酶（N〇v〇Page 13 1225490 V. Description of the invention (11) Doulton's transition membrane (HFK 131, Koch Membrane Systems, USA) is used for ultrafiltration. The hydrolysate (degree of hydrolysis between 3.8% and 4.2%) was concentrated to V C F 1 0 and the concentrated solution was directly dried. The filtrate was concentrated by evaporation to 25% solids and dried. The A C E-I measurement and flavor acceptance scores were determined as in Example 1. The ACE-I activity of the filtered powder was increased (the I Cm of the filtered powder was 0 · 15 g / litre). The original hydrolysate taken out before ultrafiltration, and its ACE-1 activity after drying was 0.43 g / liter. Both the concentrated liquid dried powder and spray-dried powder have high flavor acceptance. Example 4 Three different solutions from ALACEFM 392, ALACE Rating 421 and a mixture of ALACENTM 392 and lactose φ were made up to 5% solids water to produce 150 liters. With a commercial enzyme produced by Bacillus subtilis: neutral enzyme (Nov.

Nordisk，丹麥）及由Klyveromyce-s lactis生產之商品化 ‘ 酵素：乳糖酶（Lactozyme 30 0 0L，N0V0 Nordisk)處理溶. 溶液。酵素添加率為中性酶〇· 3%重量比（以蛋白質為基準）· 及Lactozyme 1· 2%(以乳糖酶為基準）。反應在ρΗ 7· 〇土， - 50 C下持續2小時，每〇· 5小時取出35公升樣品，以88處秒鐘使酵素失活後，進行噴霧乾燥。其活性之測疋如貫例1，二混合溶液以蛋白質為基準，分別為〇· 4公克/公升，0· 3 36公克/公升及〇· 432公克/公升。得自 ALACENTM 392之樣品的苦味度與兩個標準樣品相比較。若將苦味度分為1到10分，且以10分為苦味最強者，則水〇·5及2小時後之樣品的苦味度值分別為19及2·3，而水程度較大的兩個標準樣品苦味度值分別為5及7。 _Nordisk, Denmark) and a commercial product produced by Klyveromyce-s lactis ‘Enzyme: Lactozyme (Lactozyme 300 0 0L, NOV0 Nordisk) processing solution. The enzyme addition rate is neutral enzyme 0.3% by weight (based on protein) and Lactozyme 1.2% (based on lactase). The reaction was continued at ρ 下 7 · 〇 soil at -50 C for 2 hours. A 35 liter sample was taken every 0.5 hours, the enzyme was deactivated at 88 seconds, and then spray-dried. The activity was measured as in Example 1. The two mixed solutions were based on protein, which were 0.4 g / L, 0.336 g / L and 0.432 g / L, respectively. The bitterness of the sample obtained from ALACENTM 392 was compared with two standard samples. If the bitterness is divided into 1 to 10 points, and the strongest bitterness is divided into 10 points, the bitterness values of the samples after 0.5 and 2 hours of water are 19 and 2.3, respectively, and the larger two The bitterness values of each standard sample were 5 and 7, respectively. _

1225490 五、發明說明（12) 2小時後取出之ALACENTM 421及ALACENTM 3 92和乳糖之混合物經之樣品分別具有平均粒徑為3微米或2微米。 ALACENTM 3 92之樣品經2小時水解及如上界定之不活化後具有平均粒徑為6微米。粒徑較小的樣品具較少砂碟及白粉感。經水解之A L A C E NTM 3 9 2 /乳糖混合物混合物之溶解度於該酸鹼值範圍内為最85%。ALACENTM 3 92及ALACENTM 421樣品之溶解度大約為70%，而在pH 3· 5下，溶解度稍微下降至 6 5% ° 實例5 得自 ALACENTM 392，ALACENTM 421 及 ALACENTM 392 和乳糖之混合物之三不同溶液被製成至多為3 〇 %固形物以產生7 5 公升。酵素處理係使用如實例4吃相同條件。每隔半小時所取出之樣品稀釋至固形物含量為1 5 %。除了以實例4進行 · 熱處理。ACE- I活性測定法同實例1，其活性分別為〇 · 5 6 〇公克/公升，〇· 440公克/公升及〇· 728公克/公升。添加0 · 1 %濃度之得自實例4及實例5之樣品至1225490 V. Description of the invention (12) Samples taken from the mixture of ALACENTM 421 and ALACENTM 3 92 and lactose taken out after 2 hours have an average particle size of 3 microns or 2 microns, respectively. Samples of ALACENTM 3 92 had an average particle size of 6 microns after 2 hours of hydrolysis and inactivation as defined above. Samples with a smaller particle size have less sand and white powder. The solubility of the hydrolyzed A L A C E NTM 3 9 2 / lactose mixture mixture is at most 85% within this pH range. The solubility of the samples ALACENTM 3 92 and ALACENTM 421 is about 70%, and the solubility drops slightly to 6 5% at pH 3.5. Example 5 Three different solutions from ALACENTM 392, ALACENTM 421 and a mixture of ALACENTM 392 and lactose It is made up to 30% solids to produce 75 liters. The enzyme treatment system used the same conditions as in Example 4. Samples taken every half an hour are diluted to a solids content of 15%. Except in Example 4, the heat treatment was performed. The ACE-I activity was measured in the same manner as in Example 1. Its activity was 0.556 g / L, 0.444 g / L, and 0.728 g / L, respectively. Add samples from Example 4 and Example 5 at a concentration of 0.1% to

Bifidobacterium lactis之標準培養基中及導致菌體生長較快且該菌之最終菌體量較不添加補充劑之控制組為高。 ♦ AUCENTM 39 2，ALACENTM 421 及 ALACENTM 392 和乳糖混合物彼等二種之水解樣品之募醣（參醣或以上）含量分別為〇· 2%，2· 1% 及7. 〇%。實例6The standard medium of Bifidobacterium lactis and the growth of bacterial cells were faster and the final bacterial volume of the bacteria was higher than that of the control group without supplements. ♦ The AUCENTM 39 2, ALACENTM 421 and ALACENTM 392 and lactose mixtures of the two hydrolyzed samples had sugar content (ginseng sugar or above) of 0.2%, 2.1%, and 7.0%, respectively. Example 6

第15頁 1225490 五、發明說明（13) 以實例5中所製備之水解物粉末 ,, 甘、夫a旦几田冬作為酸礼路（yoghurt)之添加物：其：加罝佔隶終酸乳各之2 5%及5%，結果與控制組相比較，，、滑潤性增加且質地酶亦有改盖。實例7 ° 以實例5中所製備之水解物扒古从^ … w初杨末作為密斯里條食譜 (muesli bar recipe)之尽白暂水、Ε + 〈贫白貝來源，添加率為重量比6% 及1 2 %。所有的品嚐者對皮% 7欠士曰水解物條的喜好度均 WPC控制組。其中以添加由實例 ' ^ ^ w 々街a例b中所製備之經水解之 ALACENTM 42 1 及ALACENTM ^ 她 A , 和礼糖之混合物者有最好的結果。實例8 =貝例5^中所製備之水解物粉末作為代餐概念樣品之組刀。於礼糖及蛋白質中被水解7的aucentm 42ι以佔重量力:率添加至全乳粉，麥芽糊精，蔗糖及乳鈣質，配製成粉末狀代餐原型。與不含乳清蛋白生i t制組樣品相較之下，添加實例5中所製備之乳 >月蛋白水解物者之接受度較佳。實例9 ,签., 每例5呂4^ 的乳清蛋白飲料經調配而含有重量比為8%之於 ,所製備之ALACENTM 392 或 ALACENTM 421 或 ALACE, 〇 y z彳口乳糖夕、β人酸，香，合物的水解物。該飲料亦含有蔗糖，檸檬粗& 9 ;及著色劑。該飲料之酸鹼值被調整至4 · 3。該飲科马一結合5丨之軟性飲料蛋白質之營養性及健康性優點且口味清爽〃' °與含有未處理之乳清蛋白的控制組比較，Page 15 1225490 V. Description of the invention (13) The hydrolysate powder prepared in Example 5 was used, and Gan, Fu Dan and Tian Tiandong were added as yoghurt additives: its: add saccharine to the terminal acid The milk was 2 5% and 5% each. Compared with the control group, the results showed that the lubricity increased and the texture enzymes also changed. Example 7 ° The hydrolysate Bagu from ^… was prepared in Example 5 as early as the Muesli bar recipe, and the water was used as a source of ＋ + 〈poor white shellfish. 6% and 12%. All tasters' preferences for skin% 7 owe to the hydrolysate bar are WPC control group. Among them, the best results were obtained by adding the hydrolyzed ALACENTM 42 1 and ALACENTM ^ She A, which were prepared in Example b. Example 8 = The hydrolysate powder prepared in Example 5 ^ was used as a sample set for the meal replacement concept. The aucentm 42 ™, which is hydrolyzed 7 in ceremonial sugar and protein, is added to the whole milk powder, maltodextrin, sucrose, and milk calcium to make up a powder meal replacement prototype. Compared with the sample made without the whey protein and raw protein, the acceptance of the milk > moon protein hydrolysate prepared in Example 5 was better. Example 9: The amount of each whey protein beverage of 5 to 4 ^ was formulated to contain 8% by weight of the prepared ALACENTM 392 or ALACENTM 421 or ALACE, 〇yz lactose, β-human acid , Fragrant, compound hydrolysate. The drink also contains sucrose, lemon &9; and a coloring agent. The pH of the beverage was adjusted to 4 · 3. This drink Coma combines the nutritional and health benefits of 5 丨 soft drink protein with refreshing taste ° '° compared with the control group containing untreated whey protein,

第16頁 1225490 五、發明說明（14) ' 其對酸驗值之安定性有所改善且該飲料之外觀與牛乳較相像。實例1 0 另一營養性的乳清蛋白飲料被調配成含有重量比為 1 2· 5%之於實例5中所製備之ALACENTM 421水解物之水溶 ❿ /夜’在與經巴斯德式殺菌之全乳混合。添加佔最終配方已％之蔗糖及女定劑。視需要於該飲料中添加相香蕉，香草或其他類似的香料。得到延長的保存期限，將該飲料經過 1 4 0 C，3秒鐘之超高溫瞬間加熱處理。經過該額外的超高溫瞬間加熱處理後之平均粒徑仍然為3微米。實例11 以實例5中之方法進行水解，但以ALACENTM 421粉末還原液取代新鮮配製之ALACENn ，並游之濃縮成為含3〇%固形、物之溶液。中性酶之添加量改為〇 . 9 %重量比（基準），乳糖酶之用量如所界定者。2小時之m貝岸為、昆合物於固形物15%之下不活化應。ACE—丨活性為〇 48〇公克/ 。其官能性’粒徑及食品應用性均與實例4及實例5極為近似。實例1 2 以實例4中之方法進行ALACE評421粉末之水解， : '加率改：9%重量比(以蛋白質為基準)乳糖以得自米麴囷ASpergl 11US oryzae生產之乳糖酶轉變Page 16 1225490 V. Description of the invention (14) 'The stability of the acid test value has been improved and the appearance of the beverage is similar to milk. Example 10 Another nutrient whey protein beverage was formulated to contain 12.5% by weight of the ALACENTM 421 hydrolysate prepared in Example 5 in a water-soluble solution / night 'in a pasteurized solution. Of whole milk blend. Add sucrose and female fixatives which make up the final formula. Add bananas, vanilla or other similar spices to the beverage as needed. To obtain an extended shelf life, the beverage was subjected to an instantaneous heat treatment at 140 ° C for 3 seconds. The average particle size after this additional ultra-high temperature instantaneous heat treatment was still 3 microns. Example 11 The hydrolysis was carried out in the same manner as in Example 5, but the freshly prepared ALACENn was replaced with ALACENTM 421 powder reducing solution and concentrated to a solution containing 30% solids. The amount of neutral enzyme was changed to 0.9% by weight (baseline), and the amount of lactase was as defined. 2 hours of mbe is that the compound should not be activated below 15% of solids. The activity of ACE- is 0.40 g / g. Its functionality 'particle size and food applicability are very similar to those of Examples 4 and 5. Example 12 The hydrolysis of ALACE 421 powder was carried out in the same manner as in Example 4. The addition rate was changed: 9% by weight (based on protein). Lactose was converted from lactase produced by ASpergl 11US oryzae.

酶30’ 〇〇〇，Kyowa Enzymes Co. Ltd.日本）^ θ A …量比(以乳糖為基準H.5小時之後，該反添=合為 1225490 五、發明說明（15) 物以直接喷入蒸氣使溫度達8 8 °C，維持1. 5或3秒而不活化。其粒徑為2 · 3微米。官能性及食品應用性均與實例4之產物極為近似。實例1 3 以一商品化酵素：由枯草桿菌生產之中性酶（N 0 v 〇 “『(^31^’丹麥）於酵素濃度為〇.9%重量比時水解含重量比 10%之ALACENTM 392溶液。該反應於50 °C下持續6小時。每小時取出2 0 0毫升樣品，於8 8 °C下處理8秒以不活化並隨後將之冷凍乾燥。 ACE- I活性，水解度，溶液之酸鹼值及苦味度隨時間之發展如下。苦味值愈高則苦味愈強。測定值愈小則ACE_ I 活性愈高。 ^ 表1 : ALACENTM 3 92 WPC 之水解水解時間 (小時） ACE-Ι活性 (公克/公升） (IC5〇) 水解度 (%) 溶液酸驗值苦味值 (非正式的， 0-10) 1 0.420 3.86 7.01 0 2 0.280 3.78 6.96 1 3 0.230 4.53 6.92 1 4 0.220 4.89 6.89 3.5 5 0.210 5.20 6.87 2 6 0.190 5.37 6.87 4.5Enzyme 30 ′ 〇〇〇〇, Kyowa Enzymes Co. Ltd. Japan) ^ θ A… the amount ratio (based on lactose H. 5 hours, the reverse addition = 1225490 total 5. Description of the invention (15) to spray directly Into the steam to achieve a temperature of 8 8 ° C, maintaining 1.5 or 3 seconds without activation. Its particle size is 2.3 microns. Functionality and food applicability are very similar to the product of Example 4. Example 1 3 Commercial enzyme: The neutral enzyme (N 0 v 〇 "" (^ 31 ^ 'Denmark) produced by Bacillus subtilis hydrolyzes a solution containing 10% by weight of ALACENTM 392 at an enzyme concentration of 0.9% by weight. This reaction Continue for 6 hours at 50 ° C. Take out 200 ml samples per hour, treat at 8 ° C for 8 seconds to inactivate and freeze freeze dry it later. ACE-I activity, degree of hydrolysis, pH of solution The development of bitterness over time is as follows. The higher the bitterness value, the stronger the bitterness. The smaller the measured value, the higher the ACE_I activity. ^ Table 1: Hydrolysis time (hours) of ALACENTM 3 92 WPC ACE-1 activity (g / Liter) (IC50) Degree of hydrolysis (%) Bitterness value of solution acid (informal, 0-10) 1 0.420 3.86 7.01 0 2 0.280 3.78 6.96 1 3 0.230 4.53 6.92 1 4 0.220 4.89 6.89 3.5 5 0.210 5.20 6.87 2 6 0.190 5.37 6.87 4.5

第18頁 1225490 五、發明說明（16) 實例1 4 以下列商品化蛋白酶於酵素濃度為1 %重量比，5 0 °C下將含重量比1 0%之ALACENTM 39 2溶液水解1小時。再於88 °C下處理8秒以不活化並隨後將之冷凍乾燥。表2: 以不同之酵素水解酵素 ACE-Ι活性 (公克/公升) (ic5〇) 酸鹼值水解度 [%] 蛋白酶P6，中性蛋白酶，麴菌屬菌株，阿米諾酶(Amano Enzymes) 0.274 7.0 8.9 蛋白酶A，中性蛋白酶，米麴菌，阿米諾酶(Amano Enzymes) 0.443 7.0 9.2 蛋白酶Μ，酸性蛋白酶，米麴議，阿米諾酶(Amano Enzymes) (L450 4.0 7.4 胜肽酶，中性胜肽酶，米麴菌，阿米諾酶(Amano Enzymes) 0.540 7.0 6.9 中性酶，中性細菌性蛋白酶，枯草桿菌， Novo Nordisk DK 0.510 7.0 4.3 有效酶(Genancor)，酸性真菌性蛋白酶，黑麴菌，Enzyme Services Ltd. NZ 0.510 4.0 5.6 AFP2000(Genancor)，酸性真菌性蛋白酶，黑麴議，Enzyme Services Ltd. NZ 0.550 4.0 3.9 實例1 5 ACE- I胜肽類之鑑定及其活性測定Page 18 1225490 V. Description of the invention (16) Example 1 4 ALACENTM 39 2 solution containing 10% by weight was hydrolyzed for 1 hour at 50 ° C with an enzyme concentration of 1% by weight at the following commercial protease. It was then treated at 88 ° C for 8 seconds to deactivate and subsequently freeze-dried. Table 2: Hydrolyzing enzyme ACE-1 activity with different enzymes (g / L) (ic50) pH degree of hydrolysis [%] Protease P6, Neutral protease, Pleurotus strain, Amano Enzymes 0.274 7.0 8.9 Protease A, Neutral Protease, Oryzae, Amano Enzymes 0.443 7.0 9.2 Protease M, Acid Protease, Oryza, Amano Enzymes (L450 4.0 7.4 Peptidase , Neutral peptidase, Oryzae, Amano Enzymes 0.540 7.0 6.9 Neutral enzyme, Neutral bacterial protease, Bacillus subtilis, Novo Nordisk DK 0.510 7.0 4.3 Effective enzyme (Genancor), Acid fungal Protease, Black fungus, Enzyme Services Ltd. NZ 0.510 4.0 5.6 AFP2000 (Genancor), acid fungal protease, Heiye, Enzyme Services Ltd. NZ 0.550 4.0 3.9 Example 1 5 Identification and activity of ACE-I peptide Determination

第19頁 1225490 五、發明說明（17) 得自實例3之2 0 0毫克濾過物溶於0. 1%三氟醋酸（TFA)中並注入一以溶劑A(0· 1%TFA)平衡過之邱比特（Jupiter)製備性逆相HPLC管柱中（10微米，C18，22x250毫米 [Phenomenex NZ])且連接一 FPLC 系統（Pharmacia)。胜肽類隨後於0至43%之溶劑B(含0· 08%TFA之乙烯睛）梯度下以 1 0毫升/分鐘之流速於2 4 5分鐘内被沖提出來。沖提液中之胜〗太類可經由僧測2 1 4 n m之吸光值而得知。沖提液以一自動化分管收集器於每3分鐘收集一管。Page 19, 1225490 V. Description of the invention (17) 200 mg of the filtrate obtained from Example 3 was dissolved in 0.1% trifluoroacetic acid (TFA) and injected into a solvent A (0.1% TFA) equilibrated. A Jupiter preparative reverse phase HPLC column (10 micron, C18, 22x250 mm [Phenomenex NZ]) was connected to an FPLC system (Pharmacia). The peptides were subsequently flushed out at a flow rate of 10 ml / min in a gradient of 0 to 43% of solvent B (containing 0.08% TFA of vinyl acetate) at a flow rate of 10 ml / min. The victory in the extract can be determined by measuring the absorbance of 2 1 4 n m. The eluate was collected in an automated tube collector every 3 minutes.

將所收集之每一管均加以冷凍乾燥並以重量分析法測定其所存在之胜肽量。收集物使用體外分析系統（試劑得自Each collected tube was freeze-dried and the amount of peptide present was determined gravimetrically. Collections were performed using an in vitro analysis system (reagents obtained from

Sigma product 30 5- 1 0 )包括兔肺ACE及ACE-Ι活性之分析乃是利用由白兔比色計量之ACE基質：呋喃丙醯苯基丙氨醯基甘氨醯基甘胺酸 (Furylacryl〇ylphenyiaianyigiyCylglycine (FApGG)分析A C E _ 1活性。A C E可將F A P G G 其於340nm處之吸光值下降。 34Onm處之吸光值之變化將會有最高A C E抑制活性之收集管管柱中並以較緩梯度之溶劑B 之溶劑B濃度。沖提液以一自收集一管。水解得到產物F A P及G G，使得若一胜肽可抑制ACE，則於減少。將每毫克胜肽物質具區分再注入製備性逆相肝^ 沖提’即··每分鐘提高〇. 〇9% 動化分管收集器於每〇· 5分鐘Sigma product 30 5- 1 0) including rabbit lung ACE and ACE-1 activity analysis using the ACE matrix measured by colorimetry of white rabbits: furanyl phenyl alanine glycine glycine glycine (Furylacryl 〇ylphenyiaianyigiyCylglycine (FApGG) analysis of ACE _1 activity. ACE can reduce FAPGG's absorbance at 340nm. The change in absorbance at 34Onm will have the highest ACE inhibitory activity in the collection column and with a slower gradient Concentration of solvent B of solvent B. The eluent is collected by one tube. The products FAP and GG are obtained by hydrolysis, so that if a peptide can inhibit ACE, it is reduced. Each mg of peptide substance is differentiated and then injected into the preparative reverse Xianggan ^ Extraction 'ie.. .9% increase per minute. 9% of the mobilized collector is every 0.5 minutes

將所收集之每一管均以分並將其中之單一，可鑑別的區分均加以冷凍乾燥並以重析式性逆相HPLC管柱予以分析胜肽收集起來。每一被收集之量分析法測定其所存在之胜肽Each tube collected was fractionated and a single, identifiable fraction was lyophilized and analyzed on a reversible reverse-phase HPLC column. The peptides were collected. Each peptide collected was used to determine the presence of the peptide

a^2549〇五、發明說明（18)a ^ 2549〇 5. Description of the invention (18)

里。以前述之方法測定此等經純化之M 並計算個別胜肽之Κ5。。之胜肽類的ACE-丨活性每一胜狀之分子量以電子喷霧離子化質譜（^χ Μ 二重四極柱質譜儀）測定之。每—胜狀亦經由μ·之實 ^知描作出Tandem質譜而得到CAD圖譜。每一胜肽亦以自動化N端定序儀（ABI model 476A蛋白質定序儀）分析。以此三種技術之所有資料來推測所有具有ace— !活性之胜肽類。此等具有生物活性之胜肽類來源乃藉由與包含已知之所有牛乳蛋白序列的資料庫比對而得。此等胜肽類’其來源’活性及已知之相似度均列於表 3。雖然最後三個胜肽之來源為酪蛋白，但其得自乳清蛋白之水解物。用以將酪蛋白沉澱之凝乳酶並不使此等酪蛋白區分沉殿且其仍與乳清蛋白在7一起。in. These purified Ms were determined in the manner described above and the K5 of individual peptides was calculated. . ACE- 丨 activity of the peptides The molecular weight of each peptide was determined by electron spray ionization mass spectrometry (^ χ M double quadrupole mass spectrometer). Each victory also obtained Tandem mass spectrometry through the actual description of μ ·, and obtained CAD map. Each peptide was also analyzed using an automated N-terminal sequencer (ABI model 476A protein sequencer). Based on all the information of these three techniques, we can infer all the peptides with ace-! Activity. These biologically active peptide sources were obtained by alignment with a database containing all known bovine milk protein sequences. The activity of these peptides, their source, and their known similarities are shown in Table 3. Although the source of the last three peptides is casein, it is derived from the hydrolysate of whey protein. The chymosin, which is used to precipitate casein, does not distinguish these caseins from the sink and it is still with whey protein.

第21頁 1225490 五、發明說明（19) 表3. ACE-Ι胜肽類及其活性胜肽序列a 來源活性b (K;以毫克公 fh1為單位）與已知之 ACE-I胜肽之相似性類 AFE (Ala-Phe-Glu) PPd3(129-131) 20 LFSH (Leu-Phe-Ser-His) PP3(125-128) 30 ILKEKH (Ile-Leu-Lys-Glu-Lys-His) PP3(71-76) 20 LIVTQ (Leu-Ile-Val-Thr-Gln) /3-LGe(l-5) 17 MKG (Met-Lys-Gly) /3-LG(7-9) 24 LDIQKC (Leu-Asp-Ile-Gln-Lys) /3-LG(10-14) 17 /S-LG(9-14) VF (Val-Phe) /3-LG(81-82) 19 ALPMH (Ala-Leu-Pro-Met-His) ；S-LG(142-146) 12 /3-LG(142-148) VTSTAV (Val-Thr-Ser-Thr-Ala-Val) GMPf(59-64) 30 LHLPLP (Leu-His-Leu-Pro-Leu-Pro) /3-CNS(133-138) 7 LVYPFPGPIPNSLPQNIPP (Leu-Val-Tyr-Pro-Phe-Pro-Gly- Pro-Ile-Pro-Asn-Ser-Leu-Pro- Gln-Asn-Ile-Pro-Pro) /?-CN(58-76) 19 /3-CN(74-76) LFRQ (Leu-Phe-Arg-Glu) asrCN(136-139) 17h ❶Page 211225490 5. Description of the invention (19) Table 3. ACE-1 peptides and their active peptide sequences a source activity b (K; in milligrams fh1) is similar to known ACE-I peptides AFE (Ala-Phe-Glu) PPd3 (129-131) 20 LFSH (Leu-Phe-Ser-His) PP3 (125-128) 30 ILKEKH (Ile-Leu-Lys-Glu-Lys-His) PP3 ( 71-76) 20 LIVTQ (Leu-Ile-Val-Thr-Gln) / 3-LGe (l-5) 17 MKG (Met-Lys-Gly) / 3-LG (7-9) 24 LDIQKC (Leu-Asp -Ile-Gln-Lys) / 3-LG (10-14) 17 / S-LG (9-14) VF (Val-Phe) / 3-LG (81-82) 19 ALPMH (Ala-Leu-Pro- Met-His); S-LG (142-146) 12 / 3-LG (142-148) VTSTAV (Val-Thr-Ser-Thr-Ala-Val) GMPf (59-64) 30 LHLPLP (Leu-His- Leu-Pro-Leu-Pro) / 3-CNS (133-138) 7 LVYPFPGPIPNSLPQNIPP (Leu-Val-Tyr-Pro-Phe-Pro-Gly- Pro-Ile-Pro-Asn-Ser-Leu-Pro- Gln- Asn-Ile-Pro-Pro) /?-CN (58-76) 19 / 3-CN (74-76) LFRQ (Leu-Phe-Arg-Glu) asrCN (136-139) 17h ❶

第22頁Page 22

以單字母碼表示並將相對之三字母碼寫在胺基酸續列括狐中使用有色之基質：fapgg 水解物中含量最多之ACE-I 蛋白酶凍万一乳球蛋白 _巨胜肽冷—酪蛋白人另來源未知之胜肽一起測定活性It is represented by a single letter code and the corresponding three-letter code is written in the amino acid sequel. The colored matrix is used: the ACE-I protease frozen with the most content in fapgg hydrolysate in case of lactoglobulin_Jusheng peptide cold— Casein humans and peptides of unknown origin

實例1 6 =，自發性向血壓大白鼠（shr/n)進行體内血壓試驗以，例3中所製備之水解物粉末—（未經超過遽者）之功放4種大白鼠乃是經過特別之培育使其於成熟期具有高 ^且被廣泛地用來測定降血壓劑之功效。其購自動物資源中％，p 〇 Box 1180 Canning Vale ， Western Australia 6155 。Example 16 = Spontaneous blood pressure test was performed on blood pressure rats (shr / n) spontaneously. The hydrolysate powder prepared in Example 3— (not more than 遽) was used to power amplifier four kinds of rats. It is cultivated to have a high level of maturity and is widely used to determine the efficacy of antihypertensive agents. % Of its purchased animal resources, p 0 Box 1180 Canning Vale, Western Australia 6155.

於試驗中，將八週大之大白鼠個別地關在塑膠鼠籠並置於有溫控之設備中。無限制地供應其飲水並以商品化之大白鼠钥料（ad 1 ibi turn)餵養。於八週之飼養期間，每曰以一定劑量經口餵食試驗產品，並記錄其血壓變化。其血壓以一特別設計之試驗及血壓測定設備（IITC Inc·，Life Science Instruments, 23924 Victory Blvd, Woodland H i 1 1 d，C A 9 1 3 6 7 )測定之。該實驗之設計乃經梅西In experiments, eight-week-old rats were individually housed in plastic squirrel cages and placed in temperature-controlled equipment. Supply them unlimited water and feed them with a commercial rat ad 1 ibi turn. During the eight-week rearing period, the test product was orally fed at a certain dose every day, and changes in blood pressure were recorded. His blood pressure was measured with a specially designed test and blood pressure measuring device (IITC Inc., Life Science Instruments, 23924 Victory Blvd, Woodland Hi 1 1 d, CA 9 1 3 6 7). The experiment was designed by Messi

(Massey)大學動物倫理於此八週中，每組動會涊可，案號為98/1 41。 (以最小平方均值表、且物之收縮期血壓變化圖示於圖2中及4公克/公斤體重^) °飯食水解物2公克/公斤體重/日只餵食商品化之大白&心兩4組，其收縮期血壓顯著地低於 P<〇.〇〇4，見表3)。雖經最小平方均值分析，其已知之ACE抑制藥物.；;；!：解物之降血壓功效並不如-/八（蝴去/ ’、開脫曰（captopril)於施藥量30毫克 a斤胆重/日之效果，<旦對於只餵食商品化之大白鼠飼料者之血壓已有顯著性的改善。參考文獻(Massey) University Animal Ethics In each of these eight weeks, each group will be okay, case number 98/1 41. (The table shows the change in systolic blood pressure in the mean square of the least squares, and the graph of the systolic blood pressure changes in Figure 2 and 4 g / kg body weight ^) ° Meals hydrolysate 2 g / kg body weight / day Group, its systolic blood pressure was significantly lower than P < 0.04, see Table 3). Although it is analyzed by means of least squares, its known ACE inhibitory drug. ;;;!: The blood pressure-lowering effect of the solution is not as good as-/ 八（蝶去 / ', Captopril） at a dosage of 30 mg a gallbladder The effect of weight / day, < has significantly improved the blood pressure of those who fed only commercial rat feed.

Bernal V & Jelen P (1989), Effectiveness of lactose hydrolysis in Cottage- cheese whey for the development of whey drinks. Mi 1 chwissenchaft 44: 222-225Bernal V & Jelen P (1989), Effectiveness of lactose hydrolysis in Cottage- cheese whey for the development of whey drinks. Mi 1 chwissenchaft 44: 222-225

Cushman D W & Cheung H S(1971),Cushman D W & Cheung H S (1971),

Spectrophotometric assay and properties of the angiotensin converting enzyme in rabbit lung.Spectrophotometric assay and properties of the angiotensin converting enzyme in rabbit lung.

Biochem Pharmacol 20: 1637-1648. FR 2309154， 30 December 1976 Fromageries Bel La •’Biochem Pharmacol 20: 1637-1648. FR 2309154, 30 December 1976 Fromageries Bel La • ’

Vache Qui (From)， France. US 3970520, 20 July 1976, General Electric Co, USA. EP0117047, 29 August 1984, General FoodsVache Qui (From), France. US 3970520, 20 July 1976, General Electric Co, USA. EP0117047, 29 August 1984, General Foods

第24頁 1225490 五、發明說明（22)Page 24 1225490 V. Description of the invention (22)

Corporation, USA.Corporation, USA.

Maubois J L, L e on i 1 J, T r ouv e R & Bouha1 1 ab S (19 91) Les peptides du 1 a i t a act i v i t e phys i o1og i que III. Peptides du 1 a i t a effect card i ovascu1 a i re : ac t i v i t e s anti thrombo t i que e t anti hypertensive. La i t, 71， 249 -25 5. JP 4282400, 7 October 1992, Calpis ShokuhinMaubois JL, Le on i 1 J, T rouv e R & Bouha1 1 ab S (19 91) Les peptides du 1 aita act ivite phys i o1og i que III. Peptides du 1 aita effect card i ovascu1 ai re: ac tivites anti thrombo ti que et anti hypertensive. La it, 71, 249 -25 5. JP 4282400, 7 October 1992, Calpis Shokuhin

Kogyο KK, Japan· EP065663, 1 December 1982, Miles LaboratoriesKogyο KK, JapanEP065663, 1 December 1982, Miles Laboratories

Incorporated, USA. JP 8056568, 17 August 1994, Morinaga Milk Co LtdIncorporated, USA. JP 8056568, 17 August 1994, Morinaga Milk Co Ltd

Japan. EP47 45 5 0 6, 1 1 March 1 9 92, Morinaga Milk Co Ltd,Japan. EP47 45 5 0 6, 1 1 March 1 9 92, Morinaga Milk Co Ltd,

Japan. ❿Japan. ❿

Mullally Μ M, Meisel H & FitzGerald R J(1997) Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine )5 - 1 ac tog 1 obu 1 i n. Federation of European Biochemical Societies Letters, 402, 99-101.Mullally Μ M, Meisel H & FitzGerald RJ (1997) Identification of a novel angiotensin-I-converting enzyme inhibitory peptide corresponding to a tryptic fragment of bovine) 5-1 ac tog 1 obu 1 i n. Federation of European Biochemical Societies Letters , 402, 99-101.

Nakamura Y, Yamamoto N, Sakai K & Takano T ( 1 9 94 ) Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from Lactobacillus helveticus CP7 9 0. Journal of DairyNakamura Y, Yamamoto N, Sakai K & Takano T (1 9 94) Antihypertensive effect of the peptides derived from casein by an extracellular proteinase from Lactobacillus helveticus CP7 9 0. Journal of Dairy

O:\58\58905.ptd 第25頁 1225490 五、發明說明（23)O: \ 58 \ 58905.ptd Page 25 1225490 V. Description of the invention (23)

Science， 77， 917-922.Science, 77, 917-922.

Roy G( 1 9 92 ). Bitterness: reduction and inhibition. Trends in Food Science and Technology 3:85-91Roy G (1 9 92). Bitterness: reduction and inhibition. Trends in Food Science and Technology 3: 85-91

Roy G( 1 997 ). Modifying bitterness: Mechanism, ingredients and applications. Technom i c Publishers， Lancaster, UK. US 4358464, 9 September 1982, Superior DairyRoy G (1 997). Modifying bitterness: Mechanism, ingredients and applications. Technom i c Publishers, Lancaster, UK. US 4358464, 9 September 1982, Superior Dairy

Company, USA.Company, USA.

Yamamoto N ( 1 9 9 7 ). Ant i hyper tensive peptides derived from food proteins. Biopolymers 43: 129-134.Yamamoto N (1 9 9 7). Ant i hyper tensive peptides derived from food proteins. Biopolymers 43: 129-134.

第26頁Page 26

Claims

12254901225490

>、申請專利範 !〆種¥ ^”物之方法，包^工物活性之胜肽類之可溶性乳生以特徵在於…-或多種酵素水解含乳清 i)該酵素係熱不安定性蛋白酶，土 1 1 )水解作用係於溫度界於3 0 °C至7 0 t： Η 中性ΓηΓ仏其ΡΗ值為6至8‘5;當酵素為V性當蛋酵，素為時，其pH值為3· 5至5下進行，〖生蛋白酶 .* ； ^ ^ -<r> -X- . 用 i )當水解程度達到不超過i 5%時，終止該水解作 V)將一或多種酵素去活性化以終止水解 v)夕驟iv)之條件為足以溫二及水解物中。貝貝上文性之胜於該水解物中 2·根或 :乳iHi、:：圍第1項之方法，其中基質為甜乳清 3 ·根據申請專利範圍第1或2項之方法，其中該酵素自下列所組成之群：蛋白酶P6，蛋白酶A，蛋白酶M、，糸選肽酶，中性酶，有效酶（Validase)及AFP 2 0 0 0 (均如^生所定義）° °文 4. 根據申請專利範圍第1項之方法，，驟i v )包括加熱去活化。 5. 根據申請專利範圍第4項之方法，括加熱該水解物至溫度9 5 °C達1 0秒。 6 ·根據申請專利範圍第4項之方法，其中該水解作用係於低於6 5 °C之溫度下進行，而其中加熱去活化步驟於6 / ^ 其中酵素去活化步其中加熱去活化包> Application for patents! This method of ^ ^ "substances, including soluble peptides of active peptides of lactate, is characterized by ...-or more enzymes hydrolyzed containing whey i) the enzyme is a heat-labile protease Soil 1 1) Hydrolysis is at a temperature range of 30 ° C to 7 0 t: Η Neutral ΓηΓ 仏 Its PΗ value is 6 to 8'5; When the enzyme is V-type, when the egg is fermented, the The pH value is 3.5 to 5. [Protease. *; ^ ^-< r > -X-. Use i) When the degree of hydrolysis reaches no more than i 5%, stop the hydrolysis as V). Deactivation of one or more enzymes to stop the hydrolysis v) Xi iv) is sufficient to warm the second and the hydrolysate. Babe is better than the hydrolysate 2 · root or: milk iHi, :: The method according to item 1, wherein the matrix is sweet whey 3. The method according to item 1 or 2 of the scope of patent application, wherein the enzyme is composed of the group consisting of protease P6, protease A, protease M, and selected peptidase, Neutral enzyme, Validase and AFP 2 0 0 0 (all as defined by ^ Health) ° ° 4. According to the method of the scope of patent application for item 1, step iv) includes Thermal deactivation. 5. The method according to item 4 of the patent application, including heating the hydrolysate to a temperature of 95 ° C for 10 seconds. 6 · The method according to item 4 of the patent application, wherein the hydrolysis is based on The temperature is lower than 6 5 ° C, and the heating deactivation step is performed at 6 / ^ where the enzyme deactivation step is performed, and the heating deactivation step is performed.

O:\58\58905-930812.ptc 1225490 _案號88110663 年孑月曰修正_ 六、申請專利範圍至7 0 °C下進行1 0秒鐘至1 5分鐘。 7.根據申請專利範圍第4項之方法，其中該水解作用係於低於6 0 °C之溫度下進行，而其中加熱去活化步驟於6 0 t：至6 5 °C下進行1 0秒鐘至3 0分鐘。 8 .根據申請專利範圍第1或2項之方法，其中酵素去活化步驟包括轉變該含乳清蛋白基質之p Η值至該蛋白酶不具活性之pH值下。 9 .根據申請專利範圍第8項之方法，其中酵素去活化步驟包括如申請專利範圍第4 - 7項中任一項所述之加熱去活化。 1 0 .根據申請專利範圍第1或2項之方法，其中酵素去活化步驟iv)包括以具有一般可濾過1 0 - 5 0 0仟道耳頓UDa)之分子量之超過濾膜處理水解物。 1 1 .根據申請專利範圍第1或2項之方法，其中酵素於水解步驟i i )時可固定化於一鈍性支持物。 1 2 .根據申請專利範圍第1 1項之方法，其中該鈍性支持物為Roehm Eupergit，鹿角菜膠顆粒，幾丁聚醣顆粒或任何其他適合作為鈍性支持物之物質。 1 3.根據申請專利範圍第1或2項之方法，其中水解度為 3 - 5% ° 1 4.根據申請專利範圍第1項之方法，其中該基質亦含有重量比5 %或更高之乳糖。 1 5 .根據申請專利範圍第1 4項之方法，其中該乳糖含量 1 0 %或更高。O: \ 58 \ 58905-930812.ptc 1225490 _Case No. 88110663 Month, Amendment_ VI. Application for Patent Range to 70 ° C for 10 seconds to 15 minutes. 7. The method according to item 4 of the scope of patent application, wherein the hydrolysis is performed at a temperature lower than 60 ° C, and the heating deactivation step is performed at 60 t: to 65 ° C for 10 seconds Minutes to 30 minutes. 8. The method according to item 1 or 2 of the patent application scope, wherein the enzyme deactivation step includes changing the pp value of the whey protein-containing substrate to a pH value at which the protease is inactive. 9. The method according to item 8 of the patent application, wherein the enzyme deactivation step includes heat deactivation as described in any one of items 4 to 7 of the patent application. 10. The method according to item 1 or 2 of the scope of the patent application, wherein the enzyme deactivation step iv) comprises treating the hydrolysate with an ultrafiltration membrane having a molecular weight that is generally filterable from 10 to 500 Dauton (UDa). 1 1. The method according to item 1 or 2 of the scope of patent application, wherein the enzyme can be immobilized on a blunt support during the hydrolyzing step i i). 12. The method according to item 11 of the scope of patent application, wherein the blunt support is Roehm Eupergit, carrageenan particles, chitosan particles or any other material suitable as a blunt support. 1 3. Method according to item 1 or 2 of the scope of patent application, wherein the degree of hydrolysis is 3-5% ° 1 4. Method according to item 1 of the scope of patent application, wherein the substrate also contains 5% or more by weight lactose. 15. The method according to item 14 of the scope of patent application, wherein the lactose content is 10% or more.

O:\58\58905-930812.ptc 第29頁 1225490 _案號88110663 妒月曰修正_ 六、申請專利範圍 1 6 .根據申請專利範圍第1 4或1 5項之方法，其中存於基質之乳糖含量至多至重量比3 0 %。 1 7.根據申請專利範圍第1 4或1 5項之方法，其中存於基質之乳糖含量至多至重量比5 0 %。 1 8 .根據申請專利範圍第1 4或1 5項之方法，其中該基質亦以乳糖酶及/或/3 -半乳糖苷酶處理，於乳清蛋白水解前或水解期間或水解後，將乳糖水解成半乳糖及葡萄糖並合成半乳寡糖。 1 9 .根據申請專利範圍第1或2項之方法，其中所產生之乳清蛋白水解物中含有一或多種生物活性胜肽，係選自下列所組成之群：AFE，LFSH，ILKEKH，LIVTQ，MKG， LDIQK ， VF ， ALPMH ， VTSTAV ， LHLPLP ， LVYPFPGPIPNSLPQNIPP 及LFRQ 〇 2 0 . —種無苦味可溶性蛋白水解產物，其係根據申請專利範圍第1或2項方法所製得。 2 1 .根據申請專利範圍第2 0項之產物，其中該乳清蛋白之水解度3至5 %。 2 2 .根據申請專利範圍第2 0或2 1項之產物，其中該產物中之乳清蛋白平均粒徑小於3 0微米。 2 3 .根據申請專利範圍第2 2項之產物，其中該平均粒徑小於3微米。 2 4.根據申請專利範圍第2 0項之產物，其外觀實質上為白色。 2 5 .根據申請專利範圍第2 0項之產物，其亦含有半乳募O: \ 58 \ 58905-930812.ptc Page 29 1225490 _ Case No. 88110663 Amendment Month_ Sixth, the scope of patent application 16. According to the method of No. 14 or 15 of the scope of patent application, which is stored in the matrix The lactose content is at most 30% by weight. 1 7. The method according to item 14 or 15 of the scope of patent application, wherein the content of lactose in the matrix is at most 50% by weight. 18. The method according to item 14 or 15 of the scope of the patent application, wherein the substrate is also treated with lactase and / or / 3-galactosidase, and the whey protein is hydrolyzed before or during or after the hydrolysis. Lactose hydrolyzes into galactose and glucose and synthesizes galactooligosaccharides. 19. The method according to item 1 or 2 of the scope of patent application, wherein the produced whey protein hydrolysate contains one or more biologically active peptides, which are selected from the group consisting of AFE, LFSH, ILKEKH, LIVTQ , MKG, LDIQK, VF, ALPMH, VTSTAV, LHLPLP, LVYPFPGPIPNSLPQNIPP, and LFRQ 0. 0-a bitter taste-free soluble protein hydrolysate, which is prepared according to the first or second method of the scope of patent applications. 2 1. The product according to item 20 of the scope of patent application, wherein the degree of hydrolysis of the whey protein is 3 to 5%. 22. The product according to item 20 or 21 of the scope of the patent application, wherein the average particle size of the whey protein in the product is less than 30 microns. 2 3. The product according to item 22 of the scope of patent application, wherein the average particle size is less than 3 microns. 2 4. The product according to item 20 of the scope of patent application has a substantially white appearance. 25. The product according to item 20 of the scope of patent application, which also contains galactoides

O:\58\58905-930812.ptc 第30頁 1225490 _案號88110663 ^年及月日修正六、申請專利範圍糖。 2 6 ·根據申請專利範圍第2 0項之產物，其中包含一或多種生物活性胜肽，係選自下列所組成之群·· AFE，LFSH， ILKEKH ，LIVTQ ，MKG ，LDIQK ，VF ，ALPMH ，VTSTAV ， LHLPLP ，LVYPFPGPIPNSLPQNIPP 及LFRQ 。 2 7 . —種食品產品’包括根據申請專利範圍第2 〇項之乳清蛋白水解物產物。種降低收縮期血壓之醫藥組合物 28. 之根據申請專利範圍第2 0項之產物ν 2 9 .根據申請專利範圍第1或2項之方法，其另包括製備性液相層析自該水解物分離單一之胜狀。 /〇·根㈣請專利範圍第29項之方法，其'中該液相係為思相局效能液相層析接著快速液相層析。曰斤 31•根據申請專利範圍第30項之方》，其另分離之單一胜肽之生物活性。 ^ 4違其中生物活性係 3 2 ·根據申請專利範圍第3丨項之方法測試jk管收縮素轉換酵素抑制活性。 33· —種活性胜肽AFE。 / ° 34. —種活性胜肽LFSH。 35· —種活性胜肽ILKEKH。 3 6 · —種活性胜肽L I V T Q。 3 7 . —種活性胜肽Μ K G。 38. —種活性胜肽LDIQK。 3 9 · —種活性胜肽A L Ρ Μ Η。O: \ 58 \ 58905-930812.ptc Page 30 1225490 _Case No. 88110663 ^ Year and Month Date Amendment 6. Scope of Patent Application Sugar. 2 6 · The product according to item 20 of the scope of patent application, which contains one or more biologically active peptides, selected from the group consisting of: AFE, LFSH, ILKEKH, LIVTQ, MKG, LDIQK, VF, ALPMH, VTSTAV, LHLPLP, LVYPFPGPIPNSLPQNIPP and LFRQ. 27. A food product ' includes a whey protein hydrolysate product according to claim 20 of the patent application. A pharmaceutical composition for reducing systolic blood pressure 28. The product according to item 20 of the patent application ν 2 9. The method according to item 1 or 2 of the patent application scope, further comprising preparative liquid chromatography from the hydrolysis Separation of a single victory. The method of item 29 of the patent scope is requested, in which the liquid phase is a phase-effect liquid chromatography followed by fast liquid chromatography. 31. According to the formula of the 30th scope of the patent application, the biological activity of a single peptide isolated. ^ 4 Violations Among them, the biological activity is 3 2 · According to the method in the scope of patent application No. 3 丨 test jk tuberin-converting enzyme inhibitory activity. 33 · —An active peptide AFE. / ° 34. —An active peptide LFSH. 35 · — An active peptide ILKEKH. 3 6 · — An active peptide L I V T Q. 37.-An active peptide MG. 38. An active peptide LDIQK. 3 9 ·-An active peptide A L P Μ Η.

1225490 _案號 88110663 f、年乂月日__ 六、申請專利範圍 4 0 . —種活性胜肽V T S T A V。 41. 一種活性胜肽LVYPEPGPIPNSLPQNIPP。 4 2 . —種活性胜肚L F R Q。 4 3 . —種降低收縮期血壓之醫藥組合物，其包括一或多種有效量之根據申請專利範圍第3 3至4 2項中任一項之胜肽。1225490 _ Case No. 88110663 f, year, month, day __ Sixth, the scope of patent application 40.-a kind of active peptide V T S T A V. 41. An active peptide LVYPEPGPIPNSLPQNIPP. 4 2. — A kind of active L F R Q. 43. A pharmaceutical composition for reducing systolic blood pressure, comprising one or more effective amounts of a peptide according to any one of claims 33 to 42 of the scope of patent application.

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